Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers

Abstract

Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca²⁺ in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1-D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate-rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles.

Keywords: EM; lung; mucin; mucin bundle; mucus; regulated secretory pathway; secretion; submucosal gland.

Figure 1.

Assembly of the MUC5B N termini at low pH and high Ca ²⁺. A, the MUC5B monomer consists of the following domains: D1 (orange), D2 (yellow), D' (light blue), D3 (blue), D4 (light gray), CysD (red), the mucin domain (PTS, green), von Willebrand C (VWC), (C1 (medium gray) and C2 (dark gray)), and the cystine knot (CK) domain (black). The MUC5B-N recombinant protein included a His₆ tag and the D1, D2, D', and D3 domains. B, MUC5B-N was analyzed by gel filtration chromatography on a Superose 6 3.2/300 column previously equilibrated in 50 mm MES (pH 6.2), 150 mm NaCl (green) or 50 mm MES (pH 6.2) 150 mm NaCl, 4 mm CaCl₂ (blue). D means covalent MUC5B-N and Dx2 its non-covalent dimer (tetramer). mAU, milliabsorbance units. C, SDS-PAGE stained by Coomassie Blue of the purified MUC5B-N under reducing (left) and non-reducing conditions (right). Molecular mass standards marked in kilodalton are shown to the left of the gel.

Figure 2.

Single-particle analyses of MUC5B-N Dx2. A, an electron microscopy image of MUC5B-N oligomers. The sample was negatively stained in 2% uranyl acetate and imaged at room temperature in a Jeol JEM2100F electron microscope. B, MUC5B-N oligomers as 2D class averages of boxed out regions in negative stain. Side, tilted, and top views demonstrate an approximate 2-folded symmetry, as illustrated by yellow boxes. The size of each box is ∼40 nm.

Figure 3.

Single-particle analysis of MUC5B-N Dx2. A, 3D density map of the MUC5B-N oligomer with dihedral (D2) symmetry. The viewing directions are from the side parallel with the 2-fold axes and from the top. B, the Fourier shell correlation (FSC) curve between reconstructions produced by splitting the dataset in two halves. Both halves were reconstructed separately. The resolution (26 Å) for the reconstructed 3D density map was calculated from the curve at FSC = 0.143 (dotted line).

Figure 4.

3D structure of MUC5B-N Dx2. A, density map of MUC5B-N incubated at pH 6.2 with 20 mm Ca²⁺. Domains are color-marked D1, D2, and D3, with subheadings a–d for each of the four MUC5B-N molecules. The colored model is also shown rotating in two planes in Movie S1. In the structure recorded at pH 6.2 with Ca²⁺, the D1–D2 arms are turned inward and, by this, hook the two dimers together. B, comparison of the 3D structure of MUC5B-N Dx2 at pH 6.2 with 20 mm Ca²⁺ with the MUC5B-N at pH 7.4 without Ca²⁺ (the “fan”-like model to the left, from Ref. 9). The two models (left and A) are superimposed in the center and right (turned 90°).

Figure 5.

Packing of the MUC5B mucin in the intracellular storage granule and release into the gland lumen. A, MUC5B-N was analyzed by gel filtration chromatography on a Superose 6 3.2/300 column previously equilibrated in 50 mm MES (pH 6.2) and 150 mm NaCl (green) or incubated for 1 h in 50 mm MES (pH 6.2), 150 mm NaCl, and 4 mm CaCl₂ (red). MUC5B-N without calcium showed one peak corresponding to the dimer; with calcium incubation, most of the protein eluted in the void volume corresponding to high-order oligomers, suggesting heterogeneous oligomerization ((Dx2)_n). mAU, milliabsorbance units. B, MUC5B-N incubated at pH 6.2 with 20 mm Ca²⁺ and viewed by negatively stained TEM. Blue arrow, single Dx2 particle; red arrow, linear arrangement of particles ((Dx2)_n). C, proximal gland duct filled with linear material (green arrow), likely MUC5B mucin bundles (TEM). C', C at higher magnification. D, mucus strands moving through a human submucosal gland tract, frame from Movie S2. E, immunofluorescence in a paraffin section from pig trachea. Shown is MUC5B in bundles secreted from the submucosal gland (green). The staining was repeated in sections from five different pigs.

Figure 6.

The submucosal gland is the ultimate machine for shaping mucus bundles made of MUC5B. A, the remaining C-terminal parts of the MUC5B mucin, including the extended mucin domains and the C terminus, extends out from the D3 domain in two possible variants, parallel to the y axis (left) or parallel to the x axis (right). B, a possible packing of the Dx2 tetramer as 3D models inside the cell at the pH of the granule. C, the packing illustrated in B is suggested to have a better fit if each tetramer is tilted 20°, as can be suggested from the picture in Fig. 5B. D, drawing illustrating MUC5B unfolding with domains color-coded as in Fig. 1A and the effect of HCO₃⁻ flow (pH increase, Ca²⁺ detachment, pulling). D', model of MUC5B intracellular packing, modified from Ref. , with domain colors as in Fig. 1A. E, schematic of a submucosal gland. The serosal cells at the bottom of the gland are light blue and secrete chloride and bicarbonate ions to generate a flow as marked by dashed arrows. Mucus-secreting cells are more proximal to the gland opening and secrete MUC5B (green).